As cities are taking actions to reduce their carbon dioxide (CO<sub>2</sub>) emissions, the potential of urban vegetation to act as a carbon sink has received increasing attention. Carbon sequestration, however, has large uncertainties due to the highly variable and mosaic nature of urban vegetation. For land surface models to realistically estimate carbon sinks in urban areas, they need to be able to describe various urban vegetation types and their responses to environmental controls. One such model is the Surface Urban Energy and Water balance Scheme (SUEWS), which is an urban land surface model that can simulate energy, water and CO<sub>2</sub> exchanges in cities. For SUEWS to accurately simulate the biogenic CO<sub>2</sub> fluxes, it requires information on surface conductance controlling stomatal opening, maximum photosynthesis values, and parameters for soil and vegetation respiration. The main aims of this study are to provide a more comprehensive set of vegetation parameters derived from eddy covariance and chamber observations to extend the usability of SUEWS in different cities, and to evaluate the selection of the specific vegetation parameters on SUEWS model performance in two mid-latitude cities: Swindon (UK) and Minneapolis (US).</p> <p>The results highlight the importance of appropriate selection of surface conductance, maximum photosynthesis, and soil and vegetation parameters for different types of vegetation to be able to accurately simulate the biogenic and net carbon fluxes at monthly and annual level. Surface conductance parameters are most affected by soil moisture and specific humidity, rather than radiation and air temperature. The model performs better with tree surface conductance parameters (<em>R</em> = 0.62–0.70 in Swindon, <em>R</em> = 0.61–0.74 in Minneapolis) than lawn or low vegetation parameters (<em>R</em> ≤ 0.48 in Swindon, <em>R</em> ≤ 0.72 in Minneapolis), even in areas with smaller tree cover, highlighting the significance of urban trees in controlling overall stomatal conductance. The sensitivity of CO<sub>2</sub> fluxes to parameter selection is greater than that of latent heat fluxes with <em>R</em> ranging between -0.26–0.74 when compared to 0.60–0.90. Carbon balances are influenced by traffic levels, air temperatures, and rates of photosynthesis and soil respiration. Overall, the findings emphasize the necessity for accurate observations of urban vegetation to effectively parameterise urban vegetation and soil, as using inappropriate reference vegetation can lead to substantial errors in estimating biogenic exchanges.